The long term objective is to know the structure of mammalian ribosomes in sufficient detail so as to be able to provide a coherent molecular account of their function. This requires knowledge of the primary structure of the constituent nucleic acids and proteins and of the nature of their interactions, i.e. the identity of the nucleotides and of the amino acids involved and the chemistry of the association. The more proximal objectives, and the specific aims of this project, are to elucidate the structure and the function of the alpha-sarcin-ricin region of 28S rRNA. Alpha-Sarcin is a cytotoxic nuclease that inhibits protein synthesis by cleaving a single phosphodiester bond on the 3' side of G-4325 in 28S rRNA. The sequence around the alpha-sarcin site has been assiduously conserved and the hydrolysis of the phosphodiester bond there inactivates ribosomes whereas breaks at many (if not most) other sites has no affect on function. The importance of this region to ribosome integrity and function has been brought even more sharply into focus by the finding that ricin, a deadly toxin, acts by breaking a N-glycosidic bond (i.e. by a depurination) of nucleotide A-4324 in rat 28S rRNA; A-4324 is the purine adjacent to the alpha-sarcin cut site. It is hard to rationalize these findings without assigning a crucial role to the domain in ribosome function. To obtain information on the structure and function of this rRNA district, and to acquire insight into the nature of the interaction of proteins with it, we have synthesized an oligoribonucleotide (35-mer) that mimics the sequence and the structure (a stem and a loop) of the alpha-sarcin- ricin domain. We plan to use this substrate: (1) to identify the nucleotides that are required for the retention of the specificity of the hydrolysis by alpha-sarcin and for depurination (N- glycosidic cleavage_) by ricin A-chain; to determine (2) the nucleotides that are required for the binding of alpha-sarcin and ricin to the substrate - the goal is to distinguish between nucleotides necessary for association of the toxin proteins with the RNA from those that confer enzymatic specificity - if, in fact, there is a distinction; to ascertain (3) whether elongation factors EF-1 and EF-2 (or EF-Tu and EF-G) form stable complexes with the alpha-sarcin-ricin domain oligoribonucleotide and if they do to specify the individual bases that participate in the association; and, finally to assess (4) whether individual 60S subunit ribosomal proteins bind to the synthetic RNA.